Expression of phosphorylation-deficient stathmin attenuates HGF-induced EC barrier enhancement.

<p><b>A</b>: Endothelial monolayers transfected with phosphorylation-deficient stathmin (STMN-S63A) or empty vector (Em. Vec.) were stimulated with HGF (50 ng/ml). <b>A</b>: TER measurements were performed over 1.5 hrs. Bar graphs depict results of quantitative analysis of permeability data; n = 5; *P<0.05. <b>B</b>: Cortactin phosphorylation at Y⁴²¹ and tubulin acetylation at indicated time points of HGF treatment was monitored by Western blot. Probing for β-tubulin was used as a normalization control. Results are representative of three independent experiments. Bar graphs depict the quantitative densitometry analysis of western blot data; n = 4; *P <0.05, RDU: relative density units.</p

Involvement of Rac pathway in HGF-induced MT-associated signaling.

<p><b>A–D</b>: HPAEC were subjected to pretreatment with c-Met inhibitor (carboxamide 50 nM, 30 min) or knockdown of Rac1 or PAK1 as described in Methods and stimulated with HGF (50 ng/ml) for the indicated periods of time. <b>A</b>: Rac activation was determined by Rac-GTP pulldown assay. The content of activated Rac was normalized to the total Rac content in EC lysates. <b>B</b>: Time-dependent stimulation of stathmin phosphorylation and increase in tubulin acetylation was detected by western blot. <b>C</b>: Effect of preincubation with c-Met inhibitor on HGF-induced stathmin phosphorylation was evaluated by western blot with phospho-S⁶³-stathmin antibody. <b>D and E</b>: HGF-induced stathmin phosphorylation and tubulin acetylation in cells with Rac1 (<b>D</b>) and PAK1 (<b>E</b>) knockdown were evaluated by western blot. siRNA-induced target protein depletion was confirmed by membrane probing with Rac1 or PAK1 antibody. Equal protein loading in all assays was confirmed by membrane probing with β-actin antibody. <b>F</b>: Bar graphs depict the quantitative densitometry analysis of western blot data from four independent experiments; *P <0.05, RDU: relative density units.</p

Role of Rac1 in HGF-induced stimulation of peripheral MT network formation.

<p><b>A</b>: Cells grown on coverslips were transfected with non-specific RNA or Rac1-specific siRNA and stimulated with HGF (50 ng/ml, 10 min) followed by immunofluorescence staining with an antibody against β-tubulin. Bar  =  5 µm. Magnified images (insets) show details of MT structure. Results are representative of four independent experiments. <b>B</b>: Fraction of peripheral MT was quantified as described in Methods; *P<0.05; n = 4; 6 images from each experiment. <b>C</b>: Projection analysis of 20 consecutive images in control (<b>top panel</b>) and Rac1 knockdown (<b>bottom panel</b>) live cells before and after HGF treatment shows changes in GFP-EB1 track length. Bar  =  2 µm. Quantification of GFP-EB1 track length is presented on right panels. Results are representative of four independent experiments; eight cells have been inspected for each condition, in each experiment.</p

Expression of phosphorylation-deficient stathmin attenuates HGF-induced stimulation of peripheral MT network formation.

<p>Cells grown on coverslips were transfected with empty vector (Em. Vec.) or STMN-S63A and stimulated with HGF (50 ng/ml, 10 min). A: MT network was visualized by immunofluorescence staining of methanol-fixed cells with an antibody against β-tubulin. Transfected cells were detected by staining with His-tag antibody. Insets show magnified images with details of MT structure in non-transfected and STMN-S63A transfected cells. Bar  =  10 µm. Results are representative of three independent experiments. B: Bar graphs depict results of quantitative analysis of peripheral microtubules; n = 3; 10 cells from each experiment; *P<0.05.</p

HGF stimulates peripheral MT growth.

<p>HPAEC grown on coverslips were stimulated with HGF (50 ng/ml, 10 min) with or without pretreatment with c-Met inhibitor (carboxamide 50 nM, 30 min) followed by <b>A</b>: Immunofluorescence staining with an antibody against β-tubulin; <b>B</b>: Immunostaining with anti-EB1 antibody. Insets show high magnification images of cell periphery areas with microtubules or EB1-positive microtubule tips. Bar  =  5 µm. Results are representative of five independent experiments. Bar graphs depict results of quantitative analysis of peripheral microtubules (<b>A, right panel</b>) and peripheral EB1 (<b>B, right panel</b>) in methanol-fixed HPAEC; *P<0.05; n = 4; 6 images from each experiment. <b>C</b>: Live cell imaging of HPAEC expressing GFP-EB1 stimulated with HGF with or without pretreatment with c-Met inhibitor. Projection analysis of 20 consecutive images before and after HGF treatment shows changes in GFP-EB1 track length. Bar  =  2 µm. Quantification of GFP-EB1 track length is presented on right panels. Each pair of dots represents the median track length in a cell before and after thrombin treatment. Results are representative of four independent experiments; eight cells have been inspected for each condition, in each experiment.</p

The association between outpatient follow-up visits and all-cause non-elective 30-day readmissions: A retrospective observational cohort study

<div><p>Background</p><p>As an effort to reduce hospital readmissions, early follow-up visits were recommended by the Society of Hospital Medicine. However, published literature on the effect of follow-up visits is limited with mixed conclusions. Our goal here is to fully explore the relationship between follow-up visits and the all-cause non-elective 30-day readmission rate (RR) after adjusting for confounders.</p><p>Methods and results</p><p>To conduct this retrospective observational study, we extracted data for 55,378 adult inpatients from Advocate Health Care, a large, multi-hospital system serving a diverse population in a major metropolitan area. These patients were discharged to Home or Home with Home Health services between June 1, 2013 and April 30, 2015. Our findings from time-dependent Cox proportional hazard models showed that follow-up visits were significantly associated with a reduced RR (adjusted hazard ratio: 0.86; 95% CI: 0.82–0.91), but in a complicated way because the interaction between follow-up visits and a readmission risk score was significant with p-value < 0.001. Our analysis using logistic models on an adjusted data set confirmed the above findings with the following additional results. First, time matter. Follow-up visits within 2 days were associated with the greatest reduction in RR (adjusted odds ratio: 0.72; 95% CI: 0.63–0.83). Visits beyond 2 days were also associated with a reduction in RR, but the strength of the effect decreased as the time between discharge and follow-up visit increased. Second, the strength of such association varied for patients with different readmission risk scores. Patients with a risk score of 0.113, high but not extremely high risk, had the greatest reduction in RR from follow-up visits. Patients with an extremely high risk score (> 0.334) saw no RR reduction from follow-up visits. Third, a patient was much more likely to have a 2-day follow-up visit if that visit was scheduled before the patient was discharged from the hospital (30% versus < 5%).</p><p>Conclusions</p><p>Follow-up visits are associated with a reduction in readmission risk. The timing of follow-up visits can be important: beyond two days, the earlier, the better. The effect of follow-up visits is more significant for patients with a high but not extremely high risk of readmission.</p></div

Characteristics of patients in follow up visit and no follow up visit groups for logistic models (n = 30,702).

<p>Characteristics of patients in follow up visit and no follow up visit groups for logistic models (n = 30,702).</p

Joint distribution of the actual and scheduled follow-up visits (n = 65,539).

<p>Joint distribution of the actual and scheduled follow-up visits (n = 65,539).</p

Characteristics of patients with and without follow-up visits for survival models (n = 55,378).

<p>Characteristics of patients with and without follow-up visits for survival models (n = 55,378).</p

The effect of early follow-up visits on readmission risk for patients with different raw risk scores.

<p>The effect of early follow-up visits on readmission risk for patients with different raw risk scores.</p